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. 2021 Feb;10(4):e12064.
doi: 10.1002/jev2.12064. Epub 2021 Feb 16.

Molecular and functional profiling of apical versus basolateral small extracellular vesicles derived from primary human proximal tubular epithelial cells under inflammatory conditions

Xiangju Wang  1   2 , Ray Wilkinson  1   2   3   4 , Katrina Kildey  1   2 , Jacobus P J Ungerer  1   4 , Michelle M Hill  5 , Alok K Shah  5 , Ahmed Mohamed  5 , Mriga Dutt  5 , Jeffrey Molendijk  5 , Helen Healy  1   2   4 , Andrew J Kassianos  1   2   3   4
Affiliations

Molecular and functional profiling of apical versus basolateral small extracellular vesicles derived from primary human proximal tubular epithelial cells under inflammatory conditions

Xiangju Wang et al. J Extracell Vesicles. 2021 Feb.

Abstract

Proximal tubular epithelial cells (PTEC) are central players in inflammatory kidney diseases. However, the complex signalling mechanism/s via which polarized PTEC mediate disease progression are poorly understood. Small extracellular vesicles (sEV), including exosomes, are recognized as fundamental components of cellular communication and signalling courtesy of their molecular cargo (lipids, microRNA, proteins). In this study, we examined the molecular content and function of sEV secreted from the apical versus basolateral surfaces of polarized human primary PTEC under inflammatory diseased conditions. PTEC were cultured under normal and inflammatory conditions on Transwell inserts to enable separate collection and isolation of apical/basolateral sEV. Significantly increased numbers of apical and basolateral sEV were secreted under inflammatory conditions compared with equivalent normal conditions. Multi-omics analysis revealed distinct molecular profiles (lipids, microRNA, proteins) between inflammatory and normal conditions for both apical and basolateral sEV. Biological pathway analyses of significantly differentially expressed molecules associated apical inflammatory sEV with processes of cell survival and immunological disease, while basolateral inflammatory sEV were linked to pathways of immune cell trafficking and cell-to-cell signalling. In line with this mechanistic concept, functional assays demonstrated significantly increased production of chemokines (monocyte chemoattractant protein-1, interleukin-8) and immuno-regulatory cytokine interleukin-10 by peripheral blood mononuclear cells activated with basolateral sEV derived from inflammatory PTEC. We propose that the distinct molecular composition of sEV released from the apical versus basolateral membranes of human inflammatory PTEC may reflect specialized functional roles, with basolateral-derived sEV pivotal in modulating tubulointerstitial inflammatory responses observed in many immune-mediated kidney diseases. These findings provide a rationale to further evaluate these sEV-mediated inflammatory pathways as targets for biomarker and therapeutic development.

Keywords: apical; basolateral; human proximal tubular epithelial cells; inflammation; small extracellular vesicles.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Establishment of a Transwell model to examine polarized sEV production by human primary PTEC. (a) Human primary PTEC were seeded onto Transwell inserts (0.4 μm pore size) and grown to confluence. The defined medium (DM) was subsequently exchanged with fresh DM for normal control PTEC and fresh DM supplemented with 100 ng/ml IFN‐γ and 20 ng/ml TNF‐α for inflammatory PTEC and then further cultured for 72 h. PTEC culture medium was then harvested from the upper (apical) compartment and lower (basolateral) compartment for downstream sEV isolation. (b) Immunofluorescent microscopy of inflammatory PTEC monolayers on Transwell inserts (lateral projection) stained for β‐tubulin (green), MRP‐4 (red) and DAPI (blue). Scale bar represents 2 μm. One representative of three PTEC donor experiments. Equivalent staining profile was observed for normal control PTEC monolayers. (c) Immunofluorescent staining of PTEC monolayers on Transwell inserts for ZO1 (green), E‐Cadherin (red) and DAPI (blue) at time = 0 h and following 72 h culture under normal and inflammatory conditions. Scale bars represents 20 μm. One representative of two PTEC donor experiments.
FIGURE 2
FIGURE 2
Significantly elevated sEV production by human primary PTEC under inflammatory conditions. (a) Apical and basolateral sEV numbers (normalized to total particles from equivalent 1 cm2 area of confluent Transwell monolayer) produced by human primary PTEC under normal and inflammatory (IFN) culture conditions. Results represent mean ± SEM of eight individual PTEC donor experiments. *P < 0.05, **P < 0.01, Wilcoxon matched‐pairs signed‐rank test. (b) Equivalent size distribution of apical and basolateral sEV derived from primary human PTEC under normal and inflammatory (IFN) conditions; analysed with tunable resistive pulse sensing (TRPS) using a NP100 nanopore at a 45 mm stretch. Size distribution data represent the proportion of total sEV particles for each condition, with the mean ± SEM of eight individual PTEC donor experiments presented. (c) Electron microscopy images of apical and basolateral sEV purified from primary human PTEC under normal and inflammatory (IFN) culture conditions. Scale bar represents 200 nm. One representative of two PTEC donor experiments.
FIGURE 3
FIGURE 3
Human primary PTEC cultured under inflammatory conditions produce increased numbers of sEV – defined by CD63/CD9/CD81 content. (a) Western Blot for calnexin, CD63, CD9, and CD81 in PTEC lysate (2 μg total protein) and purified apical and basolateral sEV lysates (particles from equivalent 1 cm2 area of confluent PTEC monolayer) produced by human primary PTEC under normal and inflammatory (IFN) culture conditions. Representative data from eight individual PTEC donor experiments for calnexin, CD63 and CD9 and three individual PTEC donor experiments for CD81 are presented. (b‐d) Overall peak area signal for CD63 (b), CD9 (c), and CD81 (d) from purified apical and basolateral sEV lysates (particles from equivalent 1 cm2 area of confluent PTEC monolayer) produced by human primary PTEC under normal and inflammatory (IFN) culture conditions. Results represent mean ± SEM of eight individual PTEC donor experiments for CD63 and CD9 and three individual PTEC donor experiments for CD81. *P < 0.05, **P < 0.01, Wilcoxon matched‐pairs signed‐rank test.
FIGURE 4
FIGURE 4
Sixteen out of the 48 significantly differentially expressed apical sEV molecules (defined miRNA/proteins) derived from inflammatory culture conditions compared to normal culture conditions associate with a cell death and survival/immunological disease pathway identified by the “Networks Function” of IPA. The red colour intensity indicates amount of up‐regulation within the network and the green colour intensity indicates amount of down‐regulation within the network. Molecules highlighted in bold within the data table are the 16 significantly differentially apical sEV miRNA/proteins.
FIGURE 5
FIGURE 5
Eight out of 15 significantly differentially expressed basolateral sEV molecules (defined miRNA/proteins) derived from inflammatory culture conditions compared to normal culture conditions associate with a cell‐to‐cell signalling and immune cell trafficking pathway identified by the “Networks Function” of IPA. The red colour intensity indicates amount of up‐regulation within the network and the green colour intensity indicates amount of down‐regulation within the network. Molecules highlighted in bold within the data table are the eight significantly differentially basolateral sEV miRNA/proteins.
FIGURE 6
FIGURE 6
Equivalent monocyte uptake of apical and basolateral sEV derived from human primary PTEC. (a) Uptake of CFSE‐labelled, PTEC‐derived apical inflammatory sEV (green) by live (negative for LIVE/DEAD® Fixable Near‐IR Dead Cell reagent) CD3+ T cells (aqua) and CD14+ monocytes (red) as assessed by imaging flow cytometry. Equivalent images were observed for all apical and basolateral sEV populations. One representative of two PTEC donor experiments. (b) Representative flow cytometric histogram of CFSE signal by live non‐T cells/monocytes (CD3–/CD14– PBMC) (left panel), CD3+ T cells (middle panel) and live CD14+ monocytes (right panel) after culture alone (‐sEV; dashed), or following co‐culture with labelled apical inflammatory sEV in the absence (+sEV; black unfilled) or presence of cytochalasin D (+sEV+CCD; grey filled). Equivalent histograms were observed for all apical and basolateral sEV populations. One representative of five PTEC donor experiments. (c) CFSE signal (median fluorescent intensity; median FI) by live CD14+ monocytes following co‐culture with labelled apical or basolateral sEV derived from normal or inflammatory (IFN) PTEC. Results represent mean ± SEM of five individual PTEC donor experiments.
FIGURE 7
FIGURE 7
Basolateral sEV derived from human primary PTEC under inflammatory conditions induce PBMC production of chemokines (MCP‐1, IL‐8) and anti‐inflammatory cytokine IL‐10. MCP‐1 (a), IL‐8 (b) and IL‐10 (c) production by PBMC following 24 h culture alone (‐sEV) and in the presence of apical or basolateral sEV derived from normal (N) or inflammatory (IFN) PTEC. One representative donor experiment is shown (left panel). Fold change (concentration in response to basolateral sEV/concentration in response to apical sEV; basolateral/apical) in chemokine/cytokine production by PBMC for normal (N) and inflammatory (IFN) PTEC‐derived sEV is shown (right panel); Eight individual donor PTEC experiments are presented, with symbols representing individual donor PTEC experiments. *P < 0.05, Wilcoxon matched‐pairs signed‐rank test.
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